Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.     

Three-dimensional structure and function study on the active region in the extracellular ligand-binding domain of human IL-6 receptor

In this study the three-dimensional (3-D) model of the ligand-binding domain (V106-P322) of human interleukin-6 receptor (hIL-6 R) was constructed by computer-guided homology modeling technique using the crystal structure of the ligand-binding domain (K52-L251) of human growth hormone receptor (hGHR) as templet. Furthermore, the active binding region of the 3-D model of hIL-6R with the ligand (hIL-6) was predicted. In light of the structural characteristics of the active region, a hydrophobic pocket shielded by two hydrophilic residues (E115 and E505) of the region was identified by a combination of molecular modelling and the site-directed or double-site mutation of the twelve crucial residues in the ligand-binding domain of hIL-6R (V106-P322). We observed and analyzed the effects of these mutants on the spatial conformation of the pocket-like region of hIL-6 R. The results indicated that any site-directed mutation of the five Cys residues (four conservative Cys residues: Cys121, Cys132, Cys165, Cys176; near membrane Cys residue: Cys193) or each double-site mutation of the five residues in WSEWS motif of hIL-6R (V106-P322) makes the corresponding spatial conformation of the pocket region block the linkage between hIL-6 R and hIL-6. However, the influence of the site-directed mutation of Cys211 and Cys277 individually on the conformation of the pocket region benefits the interaction between hIL-6R and hIL-6. Our study suggests that the predicted hydrophobic pocket in the 3-D model of hIL-6R (V106-P322) is the critical molecular basis for the binding of hIL-6R with its ligand, and the active pocket may be used as a target for designing small hIL-6R-inhibiting molecules in our further study.     

hIL-6·hIL-6Rα·gp130三元复合物的结构预测

通过Delphi方法分析人白介素6（human interleukin-6,hIL-6）／人白介素6受体α亚基（human interleukin-6 receptor α subunit,hIL-6Rα）复合物、gp130（β subunit）的空间构象的表观静电分布,利用分子对接方法研究gp130与hIL-6/hIL-6Rα复合物作用形成三元复合物的空间构象,经过分子力学优化、分子动力学常温动态模拟借助分子间相互作用（范德华力、氢键、盐键等）、反应自由能理论探讨hIL-6·hIL-6Rα·gp130复合物稳定构象结合部位的结构域.分析结果表明,gp130蛋白表面富集较强的负电势,复合物hIL-6/hIL-6R一侧表面富集较强的正电势,gp130借助蛋白表面的静电作用结合hIL-6/hIL-6R复合物,介导hIL-6信号;hIL-6中的helix-C、loop-BC、loop-CD参与同gp130中的loopEF、linker、loopA′B′、loopB′C′、loopD′E′作用,hIL-6R中的loopA′B′、β-strand E′参与同gp130中的loopA′B′、β-strand E′作用.     

hIL-6·hIL-6Rα·gp130三元复合物的结构预测

通过Ｄｅｌｐｈｉ方法分析人白介素６（ｈｕｍａｎ ｉｎｔｅｒｌｅｕｋｉｎ－６,ｈＩＬ－６）／人白介素６受体α亚基（ｈｕｍａｎ ｉｎｔｅｒｌｅｕｋｉｎ－６ ｒｅｃｅｐｔｏｒ α ｓｕｂｕｎｉｔ,ｈＩＬ－６Ｒα）复合物、ｇｐ１３０（βｓｕｂｕｎｉｔ）的空间构象的表现静电分布,利用分子对接方法研究ｇｐ１３０和ｈＩＬ－６／ｈＩＬ－６Ｒα复合物作用成三元复合物的空间构象,经过分子力学优化、分子动力学常温动态模     

Preparation of functionally active recombinant human interleukin-6

The gene of human interleukin-6 (hIL-6) with an additional 20 amino acids on the N-end, including six histidine residues, was cloned into the expression plasmid pET28b(+). The conditions were elaborated for preparing highly active protein both using denaturing agents and without them. Application of a dialysis cascade allowed us to prepare a functionally active hIL-6 of 90-95% purity with the yield of 3 mg from liter of the cell culture. The highest activity was detected by ELISA in the preparation obtained without denaturing agents. The functional activity of hIL-6 was studied by flow cytofluorimetry. Addition of hIL-6 to the cells of immortal lines of human multiple myeloma resulted in dimerization of the gp130 receptor molecule.     

人白介素6受体结合功能域的构效关系研究

以分子对接（docking）方法研究人白介素6受体胞外区配基结合功能域“WSXWS”区氨基酸残基定点突变对受体与配基人白介素6结合时的相互作用能量、分子间相互作用的影响,从分子力学、分子动态学分析了人白介素6受体胞外区功能域关键氨基酸残基在受体与配基结合中的构象变化以及与人白介素6间的相互作用.     

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.     

The rational designed antagonist derived from the complex structure of interleukin-6 and its receptor affectively blocking interleukin-6 might be a promising treatment in multiple myeloma

Human interleukin-6 is involved in the maintenance and progression of several diseases such as multiple myeloma (MM), rheumatoid arthritis, or osteoporosis. Our previous work demonstrated that an interleukin-6 antagonist peptide (named PT) possessed potential bioactivity to antagonize the function of hIL-6 and could efficiently induce the growth arrest and apoptosis of XG-7 and M1 cells in a dose-dependent manner. In this study, the theoretical interaction of the peptide PT with its receptor was analyzed further more with molecular docking and molecular dynamics methods. The theoretical studies showed that PT possessed very high affinity to interleukin-6R and offered a practical means of imposing long-term blockade of interleukin-6 activity in vivo. According to the theoretical results, the biological evaluation of PT was researched on two different cells models with more sensitive approaches: (1) The antagonist activity of PT was studied on the interleukin-6 dependent MM cells (XG-7) cultured with interleukin-6. In the other interleukin-6 dependent MM cells (SKO-007), they survived themselves by auto/paracrine without the exogenous interleukin-6, and also could be antagonized by PT. The therapeutic value of PT only limited on the interleukin-6 dependent category in MM. (2) Myeloid leukemia M1 cells were induced for growth arrest and apoptosis in response to interleukin-6. The results supported our previous findings and showed that PT could be evaluated by protecting the cells from interleukin-6 induced apoptosis. In conclusion, PT could induce interleukin-6-dependent XG-7 and SKO-007 cells to apoptosis while inhibit interleukin-6-stimulated apoptosis in M1 cells.     

Reversal of some viral IL-6 electrostatic properties compared to IL-6 contributes to a loss of alpha receptor component recruitment

Human interleukin-6 (hIL-6) is a pleiotropic mediator of activation and proliferation across a large number of different cell types. Human herpesvirus-8 (HHV-8) has been associated with classical and AIDS-related Kaposi's sarcoma (KS). HHV-8 encodes viral IL-6 (vIL-6), a functional homolog of human interleukin-6, that promotes the growth of KS and of some lymphoma cells. Signaling induced by human IL-6 requires recruitment of the glycoprotein gp130, which acts as the signal transducing chain, and of IL-6Ralpha, which is necessary for cognate recognition and high affinity receptor complex formation. In contrast, the formation of a functional complex between vIL-6 and gp130 does not require the presence of IL-6Ralpha. The physico-chemical properties of vIL-6 have been analyzed and compared to those of hIL-6 and of the receptor chains, gp130 and IL-6Ralpha. Interaction sites on vIL-6 involve more hydrophobic residues than those of hIL-6. The electrostatic fields induced by vIL-6 and IL-6Ralpha are repulsive and prevent interaction between vIL-6 and IL-6Ralpha, whereas the electrostatic field induced by hIL-6 steers the complex formation with IL-6Ralpha. Subsequently, electrostatic binding free energy in the vIL-6/IL-6Ralpha complex is destabilizing, whereas it is stabilizing in the complex comprising hIL-6. These properties result from charge reversals between viral and human IL-6, an unusual phenomenon of amino acid substitutions within a homologous protein family. This suggests a selection pressure for vIL-6 to by-pass the IL-6Ralpha control of host defense against virus infection. This selection pressure has yielded the reversal of electrostatic properties of vIL-6 when compared to hIL-6.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.     

The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor

The members of the interleukin-6-type family of cytokines interact with receptors that have a modular structure and are built of several immunoglobulin-like and fibronectin type III-like domains. These receptors have a characteristic cytokine receptor homology region consisting of two fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 (IL-6) initially binds to its cognate alpha-receptor and subsequently to a homodimer of the signal transducer receptor gp130. The IL-6 receptor (IL-6R) consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane-proximal fibronectin-like domain (IL-6R-D3) accounts for more than 90% of the binding energy to IL-6. Here, we present the solution structure of the IL-6R-D3 domain solved by multidimensional heteronuclear NMR spectroscopy.     

Identification of the domain in the human interleukin-11 receptor that mediates ligand binding

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.     

白介素-6对NMDA诱导的小脑神经元放电活动的抑制作用及其机制

目的：观察白介素-6（IL-6）对N-甲基-D-天冬氨酸（NMDA）激发的神经元放电活动的影响及其可能的作用机制。方法：用含IL-6、NMDA和JAK抑制剂ACA90的人工脑脊液（ACSF）灌流小脑脑片,利用离体脑片神经元单位放电细胞外记录技术,记录药物对小脑间位核神经元放电的影响。用Western blot法测定间位核神经元NMDA受体亚单位1（NRI）的磷酸化水平。结果：单独用12．5μmol／L和25μmol／LNMDA灌流,神经元放电频率均较基础放电频率增加;用不同浓度IL-6（50,100,200μg／ml）联合NMDA作用后,神经尤的放电频率出现浓度依赖性地降低;AG490可部分阻断IL-6对NMDA兴奋神经元放电的抑制作用。与单独NMDA处理组比较,用IL-6联合NMDA处理神经元后,神经元的NR1磷酸化水平出现浓度依赖性地降低。AG490可阻断IL-6所致的神经元NR1磷酸化水平的降低。结论：IL-6可抑制NMDA激发的小脑间位核神经元的放电兴奋活动;并同时下调神经元的NR1磷酸化水平。     

ATP-stimulated interleukin-6 synthesis through P2Y receptors on human osteoblasts

We investigated the effect of extracellular adenosine triphosphate (ATP) on the production of interleukin (IL)-6, whose molecules are capable of stimulating the development of osteoclasts from their hematopoietic precursors as well as are involved in signal transduction systems in human osteoblastic SaM-1 cells. These human osteoblasts constitutively expressed P2X4, P2X5, P2X6, P2Y2, P2Y5, and P2Y6 purinergic receptors. ATP increased gene- and protein-expression of IL-6 in SaM-1 cells. The expression of the IL-6 mRNA was maximal at 1h, and the increase in IL-6 synthesis in response to ATP (10-100 microM) occurred in a concentration-dependent manner. Over the same concentration range of the nucleotide that was effective for IL-6 synthesis, ATP caused an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)), which increase was inhibited by pretreatment with suramin, a P2Y receptor antagonist, or 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker, but not by the extracellular Ca(2+)-chelating agent EGTA. The pretreatment of SaM-1 cells with suramin or 2-APB also inhibited the increase in IL-6 synthesis in response to ATP. These findings suggest that extracellular ATP-induced IL-6 synthesis occurs through P2Y receptors and mobilization of Ca(2+) from internal stores in human osteoblastic cells.